Novel application of fibrinogen-420 and its active domain

ABSTRACT

The invention discloses a novel application of fibrinogen-420 and its active domain (alpha EC domain), and a separate alpha EC domain protein has the same or similar function with fibrinogen-420. Fibrinogen-420 and its active domain can be widely used in inhibiting protein aggregation, helping protein refolding, drugs which can prevent and/or treat protein conformation disease, detecting denatured protein in quality control and protect protein from denaturation.

FIELD OF THE INVENTION

This invention relates to a novel application of Fibrinogen-420 and its active domain.

BACKGROUND OF THE INVENTION

Fibrinogen, also known as coagulation factor I, is an important protein in the process of blood clotting. Fibrinogen has a molecular weight of 340,000 Daltons, and is composed of two subunits connected by disulfide bonds to form dimers. Each subunit respectively consists of three intertwined polypeptide chains, called A, B, C chain. In the process of clotting, fibrinogen digested by thrombin to generate fibrin and thus form insoluble fibrin polymers. And then the composition of fibrin polymer fibers and blood platelets will form solid tampon. Fibrinogen is also a stress protein, whose content in the blood is about 1.5-4 mg/ml. The content of fibrinogen is related to the immune status, which could also reflect the risk of cardiovascular disease.

Fibrinogen-420 is a subtype of fibrinogen, in which the C-terminal of A chain has an extension of globular domain than the ordinary fibrinogen. This extension of globular domain is called the alpha EC domain, which has high homology with the globular domain at the end of B, C chains. The molecular weight of fibrinogen-420 is about 420,000 Daltons, which is different from the general tissue fibrinogen of 340,000 Dalton.

Protein misfolding disease is due to the conformational change of specific proteins in the tissue, during which proteins gather to produce amyloidosis and finally result in a class of diseases with pathological changes in tissues and organs, such as Alzheimer's disease and bovine spongiform encephalopathy. There have been no effective preventive treatments or therapeutics for these diseases by now. The existing methods such as monoclonal antibody technology, small molecules, synthetic peptides, et al. have many disadvantages including immune rejection reactions, lacking of broad-spectrum, significant side effects, and a short half-life in vivo.

There are a large number of heat shock proteins or chaperones, protect cells from high temperature, free radicals, organic solvents (eg ethanol) and other damages when the body suffers from stimulation. But the heat shock proteins do not exist in the circulatory system. The mechanism by which the body protects extracellular protein is unclear.

DESCRIPTION OF THE INVENTION

The purpose of this invention is to provide novel applications of Fibrinogen-420 and its active domain.

Our study shows that fibrinogen-420 has molecular chaperone activity and broad-spectrum, non-specific protective effects. Fibrinogen-420 is an endogenous protein, so that it will not be quickly degraded in the body and does not arouse immune rejection. It can promote denatured proteins to refold properly and stabilize protein conformation and function. So it can be widely used in protein refolding, denatured protein testing in quality control, and prevention of protein denaturation et al. The protein described could be a recombinant protein or natural protein.

Fibrinogen-420 contains alpha EC domain, and a separated alpha EC domain protein has the same or similar functions with intact fibrinogen-420. The amino acid sequence of alpha EC domain is shown as SEQ ID NO.1, and the fibrinogen-420 could specifically refer to human fibrinogen-420.

Fibrinogen-420 or alpha EC domain can be prepared into the protein reagent for use. Described protein reagents include at least fibrinogen-420 or alpha EC domain protein, and the protein reagents do not rule out other solvents and additives. Good results are expected when the ratio of other protein vs. fibrinogen-420 or alpha EC domain protein is in the range of 25:1 to 1:100, among which the ratio of 1:1 is included. The best ratio depends on the requirement of specific application.

The present invention also shows that fibrinogen-420 or alpha EC domain protein can inhibit the aggregation of degeneration protein, and protect the protein activity as well. Thus it can be used as drugs to treat protein conformation related diseases.

Wherein, the drugs to treat protein misfolding diseases contain fibrinogen-420 or alpha EC domain protein as the active ingredients. Therapeutic proteins as described can be used for the treatment of a variety of protein degenerative diseases, such as protein denaturation caused by fever, tobacco, alcohol, oxygen free radicals and other harmful substances.

The drugs for the treatment of protein misfolding diseases contain fibrinogen-420 or alpha EC domain protein as the active ingredients.

In this invention, fibrinogen-420 or alpha EC domain protein can capture the protein unfolding process. By helping the protein to refold correctly or keeping it in a folded state, fibrinogen-420 or alpha EC domain protein can prevent protein aggregation and stabilize the activity and function of a protein. Thus, fibrinogen-420 or alpha EC domain protein can enhance the ability of a protein against denaturation, so that it can be used as a protein stabilizing agent in vitro.

Wherein the protein stabilizing agent described contains fibrinogen-420 or alpha EC domain protein as the active ingredient. The protein stabilizing agent described can inhibit the precipitation of proteins which are easy to gather and form aggregation. The protein stabilizing agent can also protect the enzyme activity, such as stabilize citrate synthase, luciferase, insulin and the activity of other enzymes.

In biological diagnostic kits, particularly in ELISA immunoassay diagnostic kits, fibrinogen-420 or alpha EC domain protein can stabilize the protein reagents especially the antibodies cross-linked with reporter enzymes (such as horseradish peroxidase, alkaline phosphatase or luciferase), and increase the shelf life and the quality of the products.

Fibrinogen-420 or alpha EC domain protein can also be used to identify unfolding and denatured proteins, so it can be used in the quality control of protein reagents.

FIGURES

FIG. 1. Fibrinogen-420 or alpha EC domain protein can inhibit the thermal-induced denaturation and aggregation of citrate synthase.

FIG. 2. The alpha EC domain protein inhibits the chemical denaturation and aggregation of citrate synthase.

FIG. 3. Fibrinogen-420 inhibits the thermal-induced denaturation and inactivation of citrate synthase.

FIG. 4. The alpha EC domain protein inhibits the thermal-induced denaturation and inactivation of citrate synthase.

FIG. 5. The alpha EC domain protein specifically recognizes the denatured citrate synthase protein.

EXAMPLES Example 1 Fibrinogen-420 and Alpha EC Domain Protein Suppress Denatured Aggregation of Citrate Synthase

a. Preparation of Fibrinogen-420 and Alpha EC Domain Protein

The preparation of fibrinogen-420 started from the purification of a fibrinogen mixture from blood or cord blood, after which fibrinogen-420 can be further purified. Details are as follows:

(1) Purification of fibrinogen mixture: first, add protease inhibitors into fresh blood or cord blood, then centrifuge at 4° C. 2000 rpm and get yellow plasma from the supernatant. Then add glycine dry powder to the plasma while stirring to make glycine completely dissolved, and the final concentration of glycine is 2.1 M. After centrifugation at 5000 rpm for 15 min, the white flocculent precipitate is obtained. Dissolve the precipitate with buffer (0.15 M NaCl, 0.01 M sodium phosphate, pH 6.4 solution) that is ⅓ of the original plasma volume, and repeat this step until the dissolved volume is 1/10 of the original plasma volume. Add an equal volume of water to dilute. Then put the diluted solution at 2-5° C. for 6 hours, remove precipitate after centrifugation, add an equal volume of 0.3 M sodium chloride solution to the supernatant, then add 95% ethanol to a final concentration of 8% ethanol (volume ratio), while keep the temperature at −3 ° C., fully precipitate and the precipitation after 5000 rpm centrifugation is used as raw materials for the next step purification of fibrinogen-420. (2) Purification of fibrinogen-420: dissolve fibrinogen precipitate obtained in step (1) in 0.3 M sodium chloride solution and dialysis against 0.005 M Tris-phosphate buffer, pH8.6 (molar concentration calculated in accordance with phosphate radical). Mono Q HR 10/10 anion exchange column (Pharmacia) is used as the chromatographic column. Use pH step elution to elute the sample, starting with 0.005 M Tris-phosphate buffer rapid transition to 0.2 M Tris-phosphate buffer, pH 6.0, and then maintain the 0.2 M Tris-phosphate buffer, pH 6.0 to elute 12 column volumes, and finally eluted using a linear gradient for 12 column volumes to 0.5 M Tris-phosphate buffer, pH 4.2. Fibrinogen-420 is obtained in the last step of linear elution. The protein can be stored after dialysis against 125 mM sodium chloride, 25 mM HEPES buffer (pH 7.4). Alpha EC domain protein is obtained as follows: Alpha EC domain protein refolding and purification: Use the human liver cDNA library as a template for PCR amplification. Primers as follows:

5 -GGAATTCCATATGGACTGTGATGATGTCCTCC-3′ 5 -ACCGCTCGAGCTATTGGGTCACAAGGGGCC-3′ Restriction sites of NdeI and XhoI are introduced in the primers. The annealing temperature of PCR amplification is 55° C. Connect the αEC fragment to pET-30a expression vector (Novagen Inc.) after digested with restriction enzyme NdeI and XhoI with the same double-restriction sites. Get the recombinant bacteria after transform the recombinant expression vector into E. coli competent cells BL21/DE3 (Beijing DingGuo Biotechnology Company). Pick monoclonal recombinant bacteria to 10 ml LB (kanamycin 100 μg/ml), overnight. Then transferred to 1 liter of LB medium (kanamycin 100 μg/ml). Until the bacilli turbidity of OD600 reached 0.8 or so, add 0.5 mM IPTG-induced for 4 hours and collect bacteria by centrifugation. Collect the inclusion body protein after breaking cells. Restore the dissolved inclusion body protein, and then purify by anion-exchange column. The sample loading buffer as follows: 8M urea, 20 mM Tris-HCl and pH8.0, 30 mM BME; add 1M NaCl to loading buffer is elution buffer. Use a linear gradient elution, and collect elution peak step by step. Detect the protein purity by electrophoresis. Select the components of which purity greater than 80% to do the refolding experiments. When refolding, adjust the protein concentration to less than 0.2 mg/ml with 20 mM Tris-HCl buffer containing 8 M urea (pH 8.0), and then dialyze into 20 mM Tris-HCl, 150 mM NaCl, 1 mM chloride calcium with pH 8.0. Exchange a dialysis fluid interval of at least 4 hours, and exchange refolding solution at least for two times, fully dialysis overnight. Finally, dialyze to 20 mM Tris-HCl loading buffer for the recovery of purified protein. Use anion exchange column for purification. The sample should be centrifuged or filter with 0.22 micron pore size membrane before add to column. Use a linear gradient of salt ions, and collect protein peaks step by step. Detect the purity by oxide gel electrophoresis. b. Fibrinogen-420 and alpha EC domain protein inhibit citrate synthase thermal denaturation aggregation Citrate synthase is a key enzyme in the tricarboxylic acid cycle, but its thermal stability is poor. The temperature of 43° C. will make it denature, aggregate and precipitate. The process of citrate synthase aggregation can be examined by the changing of light scattering. The method is as follows: Detecting the process of light scattering with florescence instrument FL4500 (Hitachi, Ltd), adjusting the exciting light, emission monochromator to 500 nm and slit width to 2.5 nm Dissolving the citrate synthase in 40 nM HEPES buffer solution and making the final concentration to 0.15 μM. Simultaneously, the experiment group 1 is added with 0.15 μM fibrinogen-420, the experiment group 2 is added with 0.15 μM alpha EC domain protein, the control group 1 is added with an equal volume of HEPES buffer solution and the control group 2 is added with an equal volume of 1.2 μM bovine serum albumin Putting the samples into the 43° C. water bath and detecting the signal of light scattering. Repeat the experiment for 3 times. The light scattering signal detecting result shown in the FIG. 1 indicates that during the process of heating for 200 s the citrate synthase in the control group 1 and 2 begin to aggregate and the intensity of light scattering will be increased. However, citrate synthase aggregation in the experiment group 1 and 2 will be reduced obviously. The effect of inhibition in the experiment group 2 is better than that of group 1. Moreover, 0.15 μM alpha EC domain protein can almost totally inhibit equal molarity of citrate synthase during the thermal denaturation and aggregation process. In FIG. 1, (◯) represents control group 1, () represents control group 2, (Δ) represents experiment 1, (□) represents adding 0.6 μM alpha EC.

Example 2 Fibrinogen-420 and Alpha EC Domain Protein Protecting the Activity of Citrate Synthase (CS)

The experimental method of fibrinogen-420 and alpha EC domain protein inhibiting CS inactivation of thermal denaturation is as follows: Dissolving the citrate synthase in the 40 mM HEPES buffer solution and making the final concentration to 0.075 μM. Simultaneously, the experiment group 1 is added 0.075 μM fibrinogen-420, the experiment group 2 is added 0.15 μM fibrinogen-420, the experiment group 3 is added 0.15 μM, the experiment group 4 is added 0.15 μM alpha EC domain protein and the control group is added an equal volume of HEPES buffer solution. Putting the samples into the 43° C. water bath and beginning to detect the change of the activity of citrate synthase. The activity of citrate synthase is defined 100% before heating. The method of detecting the activity of citrate synthase is as follows:

930 μL TE buffer solution (50 nM Tris, 2 mM EDTA, pH 8.0), 10 μL 10 mM oxaloacetic acid, 10 μL 10 mM DTNB, 30 μL 5 mM acetyl-CoA.

Mixing these solutions above, adding them to the solution containing 20 μL citrate synthase quickly and detecting the dynamic change of UV absorption at 412 nm wavelength. The linearity curve slope of absorbency change represents the activity of enzyme. The determination result of the activity of citrate synthase shown in the FIGS. 6 and 7 indicates that with the time going on, the activity of citrate synthase in the control group decreases rapidly but all experiment groups can slow down the activity loss speed effectively. In FIG. 3, (◯) represents control group, (Δ) represents experiment group 1, (□) represents experiment group 2. In FIG. 4, (◯) represents control group , (Δ) represents experiment group 3, (□) represents experiment group 4.

Example 3 Alpha EC Domain Protein Recognizing Citrate Synthase Specifically

Citrate synthase and alpha EC domain protein are incubated together at 43° C. After being heated for 5 min or 10 min, antibodies of citrate synthase and alpha EC domain protein are added into the supernatant to perform co-immunoprecipitation. In the control group, citrate synthase and alpha EC domain protein are incubated together at room temperature and antibodies of citrate synthase and alpha EC domain protein are added into the supernatant to perform co-immunoprecipitation.

Results shown in the FIG. 5 indicate that after adding the antibody of citrate synthase, the denatured citrate synthase can be precipitated and alpha EC domain protein can also be precipitated at the same time. After adding the antibody of alpha EC domain protein, both of alpha EC domain protein and citrate synthase can be precipitated. The results above indicate that after being heated, citrate synthase and alpha EC domain protein can form complex so that the antibody of one protein can precipitate the other protein at the same time. In the control group, co-immunoprecipitation does not happen. The result illustrates that alpha EC domain protein can recognize and bind to the thermally denatured citrate synthase specifically.

In the FIG. 5, above of the figure is the co-immunoprecipitation of citrate synthase. Detect with antibody of alpha EC domain protein after electrophoresis. Lane 1 is the positive control. Lane 2 shows the co-immunoprecipitation after incubation for 10 min in the room temperature. Lane 3 and 4 represent co-immunoprecipitation after being heated for 5 min and 10 min respectively. Below of the figure is the co-immunoprecipitation with the antibody of alpha EC domain protein, which is detected with antibody of citrate synthase after ecectrophoresis. Lane 1 is the positive control. Lane 2 shows the co-immunoprecipitation after incubation for 10 min. Lane 3 and 4 represent co-immunoprecipitation after 5 min and 10 min of incubation respectively. In the figure, “CS” represents citrate synthase and “alpha EC” represents alpha EC domain protein. 

1. The application of inhibiting protein aggregation or refolding the denatured proteins with the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 2. According to the application of claim 1, its characteristic lies in that the molar ratio of the described a) or b) protein and denatured protein is 25:1 to 1:100.
 3. The application in preparation for drugs which can prevent and/or treat protein misfolding diseases with the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 4. One drug for preventing and/or treating protein conformation disease and its active ingredient is the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 5. The application of protein anti-denaturation with the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 6. One drug for treating protein degenerative diseases and its active ingredient is the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 7. One protein stabilizing agent whose active ingredient is the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 8. The application in quality control and detection of protein products with the following a) or b) protein; a) Its amino acid sequence is the sequence 1 in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420. 